Host range of three isolates of papaya ringspot virus (PRSV) found in North Sumatera and West Java

Papaya ringspot virus (PRSV) isolates collectd from North Sumatera and West Java were evaluated for host range using mechanical inoculation. PRSV is one of the dangerous diseases in papaya plants in Indonesia reported to have a limited host range and caused symptom variation on plants. The study aim to determine the host range of three PRSV isolates from North Sumatera and West Java to several crops. Incidence of PRSV symptoms on plants was observed by morphological characterization. The experimental results showed that two PRSV isolates from Medan Ampelas and Deli Serdang North Sumatera, and Cibinong West Java isolates were pathogenic and caused systemic symptoms to Carica papaya cv. MD (Caricacea), melon (C. melo) and bitter melon (Momordica charantia). The symptom of infected papaya by the three isolates are vein clearing, mosaic on leaves, leaf blanching, wilt, and mottle on leaf lamina. The isolates caused asymptomatic to eggplant (Solanum melongena) and choy sum (Brassica rapa), asparagus bean (Vigna unguiculata), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum syn. lycopersicum esculentum). Melon plants showed symptoms of vein clearing and lamina blanching by Medan Ampelas, and Cibinong isolates respectively, and bitter melon showed symptoms of vein clearing and leaf malformation, and mottle by isolate of Medan Ampelas and Cibinong respectively. The two plant species display asymptomatic by isolate of Deli Serdang. Isolate of Deli serdang has the potential to be a mild strain of PRSV. Complete characterization of mild strains of plant virus through molecular technology approaches can enrich understanding of the potential of these organisms for cross-protection.


Introduction
Carica papaya L., known as "Papaya", belongs to the family Caricaceae, is an economically important fruit crop grown in Brazil, Mexico, Indonesia, Philippines, Nigeria, USA, India, Jamaica, China, Taiwan, Peru, and Thailand [1].The major papaya production Indonesian districts are East Java, Central Java and West Java, Lampung, East Nusa Tenggara and followed by North Sumatra [2].Papaya production in Indonesia fluctuated year by year.In 2017 papaya production dropped to 29.172 tons [3].The fluctuating national production was one of those caused by papaya ringspot virus.
Papaya ringspot virus belongs to the family Potyviridae [4] is single stranded RNA of positive polarity having 9,000 to 10,326 nucleotides in length excluding the poly 'A' tail [5].The particle is 760-800 nm×12 nm flexuous rod [4].The virus spreads through aphids in a non-persistent manner which causes PRSV disease resulting in reduced fruit quality and yield, loss of vegetative vigor, and plant death [4].In Indonesia, this disease was first detected in Nanggroe Aceh Darussalam [6] then spread to North Sumatra, according to [7] that this disease has been found in Deli Serdang, Dairi and West Pakpak.Then it was reported the virus have spread to papaya plants in Java and Bali and even West Nusa Tenggara [8].
According to [9] that PRSV has a limited host range, which is only divided into 2 types, namely PRSV-P affects Caricaceae, Chenopodiaceae and Cucurbitaceae and PRSV-W only infects Cucurbitaceae but is unable to infect papaya.The results of the host range test conducted by [10] showed that several papaya varieties can be infected by PRSV isolates from Medan, Aceh, and Bogor.PRSV isolates from Medan and Aceh were able to infect cucumber, Japanese cucumber, watermelon, and melon, while PRSV isolate from Bogor was only able to infect cucumber, Japanese cucumber, and melon.Kabocha plants were the only plants that were not infected when inoculated with PRSV isolates from Medan, Aceh, and Bogor, while watermelon plants could not be infected by PRSV isolate Bogor.There is variation of host response on PRSV infection and study of host range are limited to a few tested isolates.Furthermore, generally viruses in plants over time will make changes either in the form of mutations or recombination in their genomic, as a strategy to adapt, survive and invade other plants if the host plant is not available.To determine the symptomatology and host range studies of PRSV isolates collected in Medan Ampelas, Deli Serdang and Cibinong in 2023 were carried out under in vivo conditions.

Plant materials
Eight plant species belonging to four families were used to identify reservoir host.The seed of eggplant (Solanum melongena), choy sum (Brassica rapa), asparagus bean (Vigna unguiculata), bitter melon (Momordica charantia), melon (Cucumis melo) and tomato (Solanum lycopersicum syn.Lycopersicum esculentum) were provided from commercial store, 3-5 leaves Nicotiana tabacum (1 month-old) was provided by a farmer in Payakumbuh West Sumatera, and 5-6 leaves Carica papaya cv.Merah Delima (MD) (15 cm in height) were purchased from a papaya farmer in Solok West Sumatra.Seed of hosts were grown 10 cm in diameter of polybag containing mixture of soil and compost (2:1 v/v) under 30±2 o C and normal daily light for at least one month, depend on different stage of the crops.

Virus sources
The virus inoculums used were isolates from PRSV infected C. papaya cv.MD from a Medan farmer's field (3 o 32ʹ13.2102"N98o 42ʹ0.2758ʺE),local papaya from farmers' fields in Deli Serdang (3 o 40ʹ31.8368ʺN98o 38ʹ32.15442ʺE)and papaya California from Cibinong (6 o 40ʹ19.8ʺS106o 45ʹ49.6ʺE) stored in silica gel.The three virus isolates have been propagated on C. papaya cv MD plants by mechanical inoculation.Symptomatic leaf tissue was grinded in a sterilized mortar and pestle in 1:20 dilution chilled phosphate buffer 0.4 g of NaH2PO4; 0.9 g of Na2HPO4, H2O, 500 ml of sterile water, pH 7.6).before sap were swabbed, leaf of healthy host plants was sprayed 600 mesh carborundum to serve as an abrasive before inoculation.Inoculated plants (each isolate) were maintained in mosquito net in Solok, West Sumatra, -(0 o 46ʹ32.52846ʺS100o 37ʹ37.17179ʺE)where daily temperature was 30±2 o C and normal daily light.The source of virus inoculum was selected from 3 leaves from top infected tissue showing primary symptoms was used due to high infective virus concentration and fewer inhibitory compounds.

Mechanical inoculation of reservoir host and papaya
The virus sap of each isolate was prepared as above method.The third-top symptomatic papaya leaf were diluted in phosphate buffer in 1:20 dilution and grinded by pestle.Third and four leaves from top of healthy host plants were applied by 600 mesh carborundum and surface of leaf were swabbed with sap of each virus isolates.Inoculated plants will be observed daily for the development of phenotypic symptoms.In this experiment, six plants of each cultivar were inoculated and evaluated up to four weeks for its local or systemic symptoms caused by viruses.

Symptom of each PRSV isolate in reservoir plants
The three PRSV isolates from Medan Ampelas, Deli Serdang and Cibinong used are PRSV isolates provided by Laboratory of Indonesia National Research and Innovation Agency.Previous studies by [8] and [11] have reported the presence of PRSV in North Sumatra (Deli Serdang and Medan Ampelas) and West Java (Bogor district) and from field observations, infected plants showed varied symptoms, but common symptoms are mosaic, vein clearing, ringspot on fruit and oily streak on the stem [8][10] [11].The three isolates have previously been propagated on C. papaya cv.MD plants 1 month-old after inoculation.In this study, the three isolates were then inoculated on C. papaya cv.MD plants that had 3-4 leaves (2 months-old).Visual observations showed that the initial symptoms detected were after 5 days of inoculated with the three isolates tested.The initial symptoms on the three isolates are in the form of discoloration on the leaves at several points and yellowing or clearing, especially on the leaf vein (Figure 1).The three isolates cause symptoms that vary and look different 4 weeks after inoculation.Phenotypic symptoms of plants affected by sap viruses from Medan Ampelas is evenly distributed clearing on leaf veins, mosaic, a little mottle, and typical oily streak on the stem, while isolate of Deli Serdang caused mild blanching, mottle and abnormal leaves.Characteristics of symptoms on papaya plants inoculated with isolates from Cibinong is symptoms of mottle leaves evenly distributed, vein clearing is evenly distributed, pale leaf colour and wilted, and transparent or typical oily streaks evenly distributed throughout the stem and even seen on the lower lamina of the leaves (Figure 1).According to [12] that mechanical inoculation of severe strains of PRSV to papaya plants caused vein clearing, typical oily streaks on the stems and stunting while the manifestation of symptoms by weak strains only resulted in subtle grey powder-like patches, which radiated from the secondary veins of young fully developed leaves.From this description, the three isolates in this test are classified as virulent strains because they cause symptoms of uniform vein clearing, mottle and oily streaks on the stem.

Symptom of each PRSV isolate in reservoir plants
In this study, eight species with each sap virus did not show specific symptoms of attack by the virus except for papaya, melon, and bitter melon plants (Figure 2, Table 1).All six plant species (tomato, melon, egg plant, asparagus bean, tobacco, and chuy sum) showed no leaf or stem deformation compared to the control plants.PRSV was described as having a limited host range.[13] PRSV from Bagalkot, Karnataka only caused limited necrosis symptoms in different quinoa (Chenopodiaceae) species, mosaic symptoms in C. sativus and asymptomatic in Phaseolus vulgaris (Fabaceae), Datura stramonium, D. metel, Capsicum annum cv.California Wonder, N. tabacum and N. glutinosa (all Solanaceae).The results of the host range test by [14] revealed that PRSV isolates from Malang also found the same, besides Caricaceae plants, PRSV isolates only caused symptoms on Cucurbitaceae and Chenopodiaceae but caused asymptomatic on B. junca and S. lycopersicum.In the present study, bitter melon infected with Medan Ampelas isolate showed symptoms of leaf blanching and malformation and Cibinong isolate caused lamina blanching, but asymptomatic by Deli Serdang isolate.While melons showed symptoms of blanching of leaf lamina after being infected with isolate from Medan Ampelas, and mottle symptoms on leaves after being inoculated with Cibinong isolate but were asymptomatic by isolate from Deli Serdang.Bitter melon and melon, a Cucurbitaceae group, are plant species commonly used as PRSV indicator plants [10][9].However, in this study, bitter melon and melon did not show symptoms after being inoculated with the Deli Serdang isolate.It is not yet certain whether the cause of the symptomless is a latent infection where the virus is dormant and exists in the cell and does not cause disease symptoms or the virus is incompatible with the host plant.For this reason, it is necessary to confirm the presence or infectivity of the three isolates on the host plant by conducting a viral genome amplification test through reverse transcriptase polymerase chain reaction (RT-PCR) technique [15], [16].
The three PRSV isolates were able to infect C. papaya cv.MD plants and cause phenotypic changes in leaves and stems.The symptoms caused were slightly different between isolates from Medan Ampelas, Deli Serdang and Cibinong as described previously with incubation period of the disease are between 5 to 6 days (Table 1).The incubation period of the three isolates is slightly shorter than previous study that stated the incubation period of the PRSV is range between 12 to 15 days [17].The daily temperature during the experiment is range between 28 to 32 o C. We suspected that the temperature condition accelerates the incubation period.The incubation period of disease by PRSV in artificially inoculated papaya plants ranges from 5-26 days in daily temperature range is 19.8ºC and 34ºC [18].In the study, the incubation period of the Medan Ampelas isolate, and Cibinong isolate are reach 15 days in melon and bitter melon.

Conclusions
Two PRSV isolates from Medan Ampelas and Deli Serdang North Sumatera, and Cibinong West Java isolates were pathogenic and caused systemic symptoms to C. papaya cv.MD (Caricacea), melon (C.melo) and bitter melon (M.charantia).The symptom of infected papaya by the three isolates are vein clearing, mosaic on leaves, leaf blanching, wilt, and mottle on leaf lamina.The isolates caused asymptomatic to eggplant (S. melongena) and choy sum (B.rapa), asparagus bean (V.unguiculata), tobacco (N.tabacum), and tomato (S. lycopersicum syn.L. esculentum).Melon plants showed symptoms of vein clearing and lamina blanching by Medan Ampelas and Cibinong isolates respectively, and bitter melon showed symptoms of vein clearing and leaf malformation, and mottle by isolate of Medan Ampelas and Cibinong respectively.The two plant species display asymptomatic by isolate of Deli Serdang.Isolate of Deli serdang has the potential to be a weak strain of PRSV.Complete characterization of plant virus strains through molecular technology approaches can enrich understanding of the potential of these organisms for cross-protection.

Table 1 .
Symptom expression of different host species after 1-month mechanical inoculation with PRSV isolates Medan ampelas isolate, 2 is Deli Serdang isolate, 3 is Cibinong isolate, vc is vein clearing, ml is leaf malformation, m is mosaic, bl is lamina blanching, w is wilting lamina, and mt is mottle.